Tyrosine hydroxylase purified to homogeneity from cultured rat pheochromocytoma cells is inactivated by incubation with its reduced pterin cofactors L-erythro- tetrahydrobiopterin, 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropterin and 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropterin. Each of the two diastereoisomers of L-erythro-tetrahydrobiopterin inactivates tyrosine hydroxylase but the natural (6R) form is much more potent than the unnatural (6S) form at equimolar concentrations. The pterin analog 6-methyl-5-deaza-tetrahydropterin, which has no cofactor activity with tyrosine hydroxylase, also inactivates the enzyme whereas the oxidized pterins 7,8 dihydrobiopterin and biopterin do not. The inactivation process is both temperature and time dependent and results in a reduction of the Vmax for both tetrahydrobiopterin and tyrosine. Neither tyrosine nor oxygen inactivates tyrosine hydroxylase. These data indicate that the binding of BH4 to tyrosine hydroxylase, in the absence of tyrosine, reduces the catalytic potential of the enzyme by changing its conformation.